First report of

Ornamental fish are becoming increasingly popular, but the lack of knowledge regarding their various diseases is a major challenge. Skin diseases commonly found in freshwater fish include black spot disease (BSD), which is characterized by melanin deposits around the metacercariae of some trematode species. Since BSD remains poorly understood, this study describes an outbreak of BSD in Etroplus maculatus raised in outdoor ponds at a Brazilian fish farm. Metacercariae samples were collected, examined, and subjected to molecular phylogenetic analysis. The parasites were conspecific to an unnamed species, Crassiphiala lineage 5, recently found in Brazilian birds (Megaceryle torquata). Sequences obtained for longifurcate cercariae of the planorbid snail Biomphalaria straminea from the same region were identical to our metacercariae of Crassiphiala sp. These results suggest that Biompahalaria snails are likely an intermediate host of this parasite on farms where E. maculatus was found to be infected. We provide the first molecular evidence that Crassiphiala are the causative agents of BSD in fish from Brazil. Combatting snails and preventing access of fish-eating birds to outdoor ponds are strategies to control this disease in ornamental fish farms.


Introduction
The aquaculture industry in Brazil focuses mainly on fish products for human consumption. However, the pet industry is growing steadily, and ornamental fish are now the fourth most popular pet in the country (Rezende et al. 2021;Valenti et al. 2021). Freshwater ornamental fish farming occurs mainly in southeastern Brazil, especially in the states of São Paulo and Minas Gerais (Faria 2016;Valenti et al. 2021).
Among the diseases commonly reported in freshwater fish are black spot disease (BSD), which is mainly caused by parasitism by members of the family Diplostomidae (Lane and Morris 2000;Niewiadomska 2002), but also by some species of Heterophyidae (Sándor et al. 2017;Denis et al. 2019;Duflot et al. 2021). The name of this disease alludes to the macroscopic aspect of infected animals, where melanin produced by melanomacrophages is deposited around the encysted metacercariae. (Bush et al. 2001;Thatcher 2006;Denis et al. 2019). This alteration also results in an unpleasant appearance of fish reared for ornamental purposes and contributes to rejection by consumers, which can be detrimental to businesses (Lane and Morris 2000).
We aimed to identify the etiological agent of BSD in Etroplus maculatus, a farmed ornamental fish species in Brazil.

Fish sampling
An outbreak of BSD was observed in E. maculatus raised in outdoor tanks at a fish farm located in the state of Minas Gerais, Brazil, in April 2019. A total of 13 live fish (mean Section Editor: Guillermo Salgado-Maldonado weight of 3 g) were collected and immediately transported to diagnostic laboratory. Fish were kept in aquaria, fed commercial fish food, and processed for parasitological analysis.

Parasitological examinations
For metacercariae recovery, the fish were euthanized by immersion in a benzocaine solution (250 mg/L). The fish integuments (skin and fins) were examined under a stereomicroscope, the metacercariae were counted, and cysts were removed using metal needles. These were mounted between glass and coverslips. Larvae were mechanically excysted and mounted as described above or killed in 70 °C water, and fixed in 10% formalin. Specimens were stained with alum acetocarmine, dehydrated in a crescent ethanol series, diaphanized in beechwood creosote, and mounted between slides and coverslips with Canada balsam. Parasite preparations were examined under a light microscope (Leica DM500) and photographed using a Leica ICC50 HD digital camera. The dimensions of the cyst and larvae were obtained from the photographs and represented in micrometers (μm). Samples of the studied parasites were deposited at the collection of trematodes of UFMG (UFMG-TRE 132).
We attempted to obtain the adult stage of the parasite for specific taxonomic identification by orally inoculating four young chickens (Gallus domesticus) and two jirds (Meriones unguiculatus). They were forced to swallow a solution containing 20 encysted metacercariae removed from E. maculata. The vertebrates were kept under laboratory conditions with ad libitum access to water and food. Fecal samples were examined using the spontaneous sedimentation technique to evaluate infection success.
Animals were euthanized and necropsied 12 days postinfection to detect adult parasites in their intestines. The use of vertebrate animals in the experiments followed the protocol approved by the Local Ethics Committee in Animal Experimentation (CEUA-UFMG, protocol 68/2017).

Molecular analyses
Samples of encysted metacercariae were fixed in ethanol 95% and stored at −20 °C until use. Partial regions of the 28S (primers Dig12 and 1500R), ITS (primers D1 and D2), and cox1 (primers JB3 and COI-R Trema) genes were amplified by PCR, using previously described conditions (Galazzo et al. 2002;Tkach et al. 2003;Miura et al. 2005). Additionally, cercariae previously found in Biomphalaria straminea in the same geographic area Sequence data were edited using Chromas Pro software (Technelysium Pty Ltd, Australia), and the contigs were used for similarity searches in the Basic Alignment Search Tool (BLAST). Alignments were constructed using MEGA X (Kumar et al. 2016), and sequences of the closest genera available in GenBank were included. The evolutionary models used in the phylogenetic analysis were determined using the Bayesian information criterion in MEGA X and outgroup selections were based on phylogenies published by Achatz et al. (2019). Phylogenetic analyses were performed using maximum likelihood and Bayesian inference methods. The maximum likelihood trees were generated in MEGA X (bootstrap test with 1000 repetitions). Bayesian inference analyses were performed in MrBayes v.3.2.6 (Ronquist et al. 2012) using Markov Monte Carlo chain searches in two simultaneous runs of four chains per 1,000,000 generations and sampling every 100 generations. The first 25% of the examined trees were discarded as "burn-in." The generated sequences were deposited at GenBank [accession numbers: OQ341467 (28S). OQ341469 (ITS), OQ346404, and OQ346405 (cox1)].

Results and discussion
Morphological and molecular data on metacercariae involved in BSD in E. maculatus revealed the presence of a species of the genus Crassiphiala Van Haitsma, 1925 (Diplostomidae: Crassiphialinae). A total of 144 metacercariae were counted in the 13 specimens evaluated, with a mean infection intensity of 11 ± 8 (3-32) metacercariae/fish. Macroscopically, cysts were found in the teguments and fins (Fig. 1), and they were covered by a dark melanin deposit produced by melanomacrophages. The resulting black spots were 0.5 mm in size.
Sequences of 28S (1227 bp), ITS (1200 bp), and mitochondrial cox1 (799 bp) were obtained for E. maculatus metacercariae. High identity with Crassiphiala was verified, 97.6-99.8% using the 28S gene. Phylogenetic analysis of 28S and ITS sequences revealed that the parasite grouped in well-supported clades containing Crassiphiala spp. (Fig. 4). Based on 28S, 100% similarity was found with Crassiphiala sp. lineage 5 (MN200261). This sequence information was recovered from Megaceryle torquata in the Brazilian Analysis of cox1 resulted in a trimmed alignment containing 372 bp and two species/lineages of the genus Crassiphiala. New sequences were also obtained for the same region of cox1 in a longifurcate cercaria (Strigeid cercaria) previously reported in B. straminea from the same geographical area (López-Hernández et al. 2019). Phylogenetic analysis revealed both larval isolates were from the same clade as Crassiphiala sp. lineage 5 (Achatz et al. 2019). The combination of this information suggests that the isolates of Crassiphiala are conspecific. However, the species differs from Crassiphiala sp. lineage 2 (molecular similarity: 92.6-93.6%). Comparison of cox1 sequences of the barcode region (for cercariae from B. straminea) confirmed the link with Crassiphiala sp. lineage 5 (96.8-97.0% similarity with M. torquata isolates). Relatively low similarities (82.7-87.7%) were found with the other four lineages/ species of Crassiphiala. Our data suggest that an unnamed species linked to Crassiphiala sp. lineage 5 is involved in cases of BSD in E. maculata. Crassiphiala is a monotypic genus of diplostomids found, when in the adult stage, in the intestine of kingfishers (Achatz et al. 2019). Recent molecular studies revealed the existence of new lineages/species of these trematodes, including two lineages found in M. torquata in South America (Achatz et al. 2019). The complete life cycle is known only for Crassiphiala bulboglossa. Adult parasites were found in kingfishers, and neascus-like melanized metacercariae were found encysted in the fish skin (Hoffman 1956).
The involvement of diplostomid species in fish BSD around different parts of the world is well known (Thatcher 2006;Flores-Lopes 2014;Barrilli et al. 2021 Kohl et al. 2019;Charo-Karisa et al. 2021;Duft et al. 2021). This disease is characterized by parasitic invasion and encysting in various fish organs, usually, the skin and muscle, where they are externally visible as black spots (Hoffman 1955;Achatz et al. 2019). In the present study, the involvement of one of these undescribed species as causative agents of BSD in ornamental fish from Brazil is reported for the first time. Specific identification of this parasite requires detailed morphological characterization of the adult stages for a formal taxonomic description as a new species of Crassiphiala. We attempted to obtain the adult stage, but no worms were found in the young chickens tested. Future helminthological studies are required to identify the adult stage of the parasite in E. maculatus.
Our study identified larval stages that were previously found in B. straminea from the same geographical area, and identified them as Crassiphialinae gen. sp. (López-Hernández et al. 2019); they were molecularly linked to the metacercariae found in E. maculatus. This planorbid snail is widely distributed and adapted to human-made aquatic environments. Their presence in outdoor ponds represents a risk for BSD and other fish diseases caused by trematodes that infect piscivorous birds. Field studies on fish farms are needed to definitively determine the involvement of this snail as a vector of Crassiphiala. For sure, the control and prevention of BSD in ornamental fish farms involved the combat of snails in the ponds. On the other hand, to prevent the access of the potential wild avian definitive host, not only kingfishers but also other groups (herons, cormorants, grebes) to the ponds can be an essential strategy to prevent the contamination of aquatic environments with parasite eggs present in feces of infected birds. Both strategies can be helpful to prevent the establishment and maintenance of the parasite life cycle and so to avoid BSD outbreaks in ornamental fish framings.